AIM2 inflammasome regulated by the IFN‐γ/JAK2/STAT1 pathway promotes activation and pyroptosis of monocytes in Coronary Artery Disease

Abstract Background Numerous studies have demonstrated that Absent in Melanoma 2 (AIM2) is upregulated in aortic plaques, especially in Vascular Smooth Muscle Cells in Coronary Artery Disease (CAD), and is related to inflammasome‐induced inflammation. However, the underlying mechanism of this phenomenon and the role of AIM2 in atherosclerosis remained unclear. Methods This study enrolled 133 CAD patients and 123 controls. We isolated Peripheral Blood Leukocytes (PBLs) and the mRNA expression of AIM2 inflammasome and its downstream genes (ASC, Caspase‐1, IL‐1β, and IL‐18) were detected by real‐time quantitative PCR (qPCR). We assessed correlations between AIM2 expressions and clinical characteristics by multiple linear regression and spearman's correlation. The THP‐1 cells cultured in poly(dA:dT), A151, interferon‐gamma (IFN‐γ), AG490, or JC2‐11. And then the mRNA and protein levels of AIM2, ASC, Caspase‐1, IL‐1β, IL‐18, GSDMD, and STAT1 were analyzed by qPCR and Western blot analysis, respectively. The migration and adhesive capacity of THP‐1 cells was assessed using an inverted microscope and an inverted fluorescence microscope, respectively. Results In this study, we found that expressions of components of AIM2 inflammasome and its downstream genes (ASC, Caspase‐1, IL‐1β, and IL‐18), were all increased in PBLs of CAD patients, which indicated the inflammasome activation. AIM2 inflammasome activation further induced pyroptosis, and stimulated migration and adhesion in monocyte cell lines, which was regulated by IFN‐γ probably through JAK2/STAT1 pathway. In addition, AIM2 expressions were positively correlated with systemic inflammatory indicators as an independent risk factor for CAD. Conclusions In conclusion, increased AIM2 expression, induced by the IFN‐γ/JAK2/STAT1 signal, orientates monocytes to inflammatory status or even pyroptosis through AIM2 inflammasome activation, which is involved in the development of CAD.


| INTRODUCTION
Coronary Artery Disease (CAD) has become a major cause of death in modern society. 1 Despite advances in prevention and treatment of CAD, it remains a threat to human health worldwide mostly due to the Residual Inflammation Risk. 2 Canakinumab Anti-Inflammatory Thrombosis Outcome Study (CANTOS) showed that it was able to stabilize plaque well and prevent the occurrence of Cardiovascular (CV) events when blocking the inflammatory process. 3Monocytes are one of the most critical immunology cells in the chronic inflammatory process of atherosclerosis. 4,5In addition, immunology cells including T cells, mast cells, dendritic cells, macrophages, and so forth.are all involved in this process, by amplifying the production and signaling of cytokines that regulate plaque formation and growth, for example, by producing large amounts of Interferongamma (IFN-γ) and Tumor Necrosis Factor-α (TNF-α). 6,7FN-γ was found to activate Absent in Melanoma 2 (AIM2) inflammasome in human keratinocytes through the JAK2-STAT1 pathway, causing a downstream inflammatory response that leads to inflammatory skin diseases. 8Whether IFN-γ could activate AIM2 inflammasome in monocytes to induce inflammation in CAD need further investigation.
AIM2 is a member of the hematopoietic IFN inducible nuclear protein family, containing a 200amino acid repeat (HIN-200).It can recognize various endogenous and exogenous Double-Strand DNA (dsDNA) that can trigger inflammasome activation.Upon activation, AIM2 starts to assemble the inflammasome complex, consisting of pro-caspase-1 and apoptosis associated speck-like protein containing a CARD (ASC).Once assembled, the AIM2 inflammasome can activate caspase-1, promote the production of IL-1β and IL-18 and induce GSDMD-mediated cell pyroptosis. 9,10Sustained activation of AIM2 inflammasome leads to inflammatory damage of tissues. 11Recent in vivo studies suggest that AIM2 inflammasome plays a role in various CV events. 12,13For example, Pan et al. found that Vascular Smooth Muscle Cells respond to inflammatory signals by upregulating the expression of AIM2. 14ecently, we found the AIM2 gene was significantly overexpressed in the CAD group in GSE42148 of Peripheral Blood Leucocytes (PBLs) in the GEO database which included 11 controls and 11 CAD patients (Supporting Information S1: Figure 1).We hypothesized that overexpressed AIM2 is probably regulated by IFN-γ/ JAK2/STAT1 pathway, and played an essential role in the inflammatory responses and activation of monocytes.So, we executed the following study to explore the function of AIM2 in monocyte inflammatory in CAD.

| Clinical cohorts
Two hundred fifty-six study subjects, including 133 CAD patients and 123 controls, were recruited at Zhongnan Hospital of Wuhan University (Wuhan, China).All subjects were unrelated Chinese Han individuals, whose age and gender were matched between patients and controls.The inclusion and exclusion criteria were consistent with previous studies. 15The clinical information of all subjects, including age, sex, medical histories, routine blood parameters, biochemical indices, and other clinical data, was collected.And, the diagnostic criteria for hypertension, hyperlipidemia and Type 2 Diabetes (T2DM) were also followed our previous study. 16The present study was authorized by the Medical Ethics Committee of Zhongnan Hospital of Wuhan University (2020195) and followed the Declaration of Helsinki.

| Isolation of primary monocytes
Sorting monocyte assay was performed using a magnetic separation column (Miltenyibiotec).After collection of 7 mL of peripheral blood from volunteers, Peripheral Blood Mononuclear Cells (PBMC) were separated by standard density gradient centrifugation. 17Then 80 μL of magnetic bead separation buffer (Miltenyibiotec) and 20 μL of magnetic beads coated with CD14 antibody (eBioscience) were added to 1 × 10 7 PBMC.The cells separated from the magnetic beads were obtained as monocytes, according to the instructions.

| RNA extraction and real-time quantitative PCR (qPCR)
The total RNA of cells was extracted using TRIzol reagent (Invitrogen) following the manufacturer's instructions.The RNA was reverse-transcribed into cDNA by PrimeScriptTM RT reagent Kit with gDNA Remover (Takara).Real-time qPCR was performed using SYBR Prime Script qPCR kit (CWBIO), specific primers on Bio-Rad CFX 96 real-time system (Biorad).The primer sequences are shown in Supporting Information S1: Table 1.The relative expression was calculated using the comparative crossing threshold cycle threshold Ct (2 C −ΔΔ t ) method with GADPH as the internal reference control.

| Protein extraction and Western blot analysis
The total protein of cells was extracted using a protein lysis solution (Beyotime).Each well was spiked with | 3 of 14 15 μg of total protein, separated by 10% SDSpolyacrylamide gel electrophoresis (SDS-PAGE).The protein bands were transferred to the PVDF membrane, then blocked with 5% bovine serum albumin for 2 h at room temperature, before incubation at 4°C overnight with primary antibody dilutions of AIM2, caspase-1, GSDMD or p-STAT1, followed by incubation with secondary antibody dilutions.The PVDF membrane was developed by ECL luminol (eBioscience).The grayscale values of the specific bands were analyzed using ImageJ software.

| Transwell assay
The migration assay was performed using Transwell insert plates (Corning).About 200 μL of serum-free RPMI 1640 medium containing approximately 5 × 10 5 cells are added to the upper chamber, while 600 μL of RPMI 1640 medium containing 200 ng/mL monocyte chemoattractant protein-1 and 5% FBS was added to the lower chamber.The chambers were incubated for 90 min at 37°C in an incubator with 5% CO 2 .The culture medium was discarded and the chambers were fixed in 800 μL of 4% paraformaldehyde (Biosharp, Beijing, China) for 10 min at room temperature, followed by the addition of crystal violet staining solution (Beyotime).Then several random fields were chosed and photographed under an inverted microscope at a magnification of ×100.The number of THP-1 cells in each area was calculated using the ImageJ software.

| Adhesion assay
The THP-1 cells were incubated with the BCECF-AM fluorescent probe (Beyotime) for 30 min in a CO 2 incubator hidden from light, washed with PBS to remove the excess probe, resuspended in serum-free 1640 medium, and added to fully adherent fused HUVEC in 12-well plates at a density of 5 × 10 5 cells/mL for 1 h.The nonadherent THP-1 cells were washed away with PBS.THP-1 cells were observed under an inverted fluorescence microscope at a magnification of ×100, and several randomly selected areas were photographed.The number of THP-1 cells in each area was calculated using the ImageJ software.3 | RESULTS

| Clinical characteristics
The basic characteristics of all participants are shown in Table 1.CAD patients and controls were matched for age and sex (p = 0.063, 0.392).CAD patients had a higher prevalence of hypertension, hyperlipidemia and T2DM than controls (p = 9.40E-09, 2.17E-20, 0.001).Also, TG and FPG were higher but HDL-C levels were lower in patients than controls (p < 0.0001).Compared with controls, the percentage of neutrophils and monocytes was significantly increased in CAD patients, while the rate of lymphocytes was significantly decreased.We also randomly selected 25−25 age-sex matched subgroups among them for further investigation on the downstream signaling pathway.The clinical characteristics of these individuals in subgroups are shown in Table 2.

| High expression of AIM2 is an independent risk factor for CAD
To investigate the association between the AIM2 expression and CAD, we divided 256 subjects to the high and low AIM2 expression subgroups according to the median of AIM2 expressions and performed a logistic regression analysis of AIM2 expressions and the prevalence of CAD.

| AIM2 inflammasome promotes inflammatory responses and causes pyroptosis in human monocyte cell lines
To reveal the biological significance of AIM2 upregulation, we investigated functions of AIM2 inflammasome in human monocyte-macrophage lines.The AIM2 inflammasome agonist poly (dA:dT) works by binding to AIM2 molecules and causing AIM2 inflammasome assembly, 18 whereas the inhibitor A151 works by competitively binding to AIM2 molecules and blocking AIM2 interaction with dsDNA thereby to hinder the assembly of inflammasome. 19s shown in Figure 3A−D, expressions of AIM2, ASC and caspase-1, and inflammatory factors (IL-1β and IL-18), and cell pyroptosis marker GSDMD were all increased, after the addition of poly(dA:dT).In contrast, the expressions of these genes decreased after the addition of A151.The same phenomenon was also observed in macrophages derived from THP-1 monocytes, primary monocytes and their derived macrophages (Supporting Information S1: Figure 2).Compared with the control, the numbers of adhered and migrated THP-1 cells were significantly increased after poly(dA:dT) treatments (Figure 3E,G).In contrast, A151 inhibited the adhesion and migration of THP-1 cells (Figure 3F,H).

| IFN-γ-induced activation of AIM2 inflammasome by the JAK2/STAT1 pathway
STAT1 protein is an important cytoplasmic transcription factor that can be induced by JAK2 phosphorylation in the presence of IFN-γ. 22Consistent with others, 23 we also found that IFN-γ-treated cells showed increased mRNA expressions of JAK2 and STAT1 (Figure 5A,C) and the protein expression of p-STAT1 (Figure 5B,D).This suggests the activation of JAK2/STAT1 pathway.
JAK2 inhibitor AG490 was reported to achieve a blocking effect on the JAK2/STAT1 pathway, 8,24 so we pretreated THP-1 cells with AG490 for 2 h, and then treated with the IFN-γ for 24 h.The results showed that JAK2/STAT1 expressions were significantly reduced, as well as the phosphorylation of STAT1 protein, in the IFN-γ + AG490 group.In addition, the addition of AG490 further reduced the expressions of AIM2 and its downstream molecules (Figure 5E,F).The results suggest that the IFN-γ/JAK2/STAT1 pathway may be the upstream signaling molecules of AIM2 inflammasome.
F I G U R E 5 (See caption on next page).

| DISCUSSION
More and more experimental and clinical evidence suggest a chronic inflammation was participated in AS diseases, in which immune cells and various inflammatory factors play an important role. 25,26Our study found that the high expression of AIM2 was an independent risk factor for CAD, that could promote inflammatory responses and pyroptosis in monocyte, which might be regulated by the IFN-γ/JAK2/STAT1 pathway. 12searchers first observed a significant increase of AIM2 in the necrotic core of AS plaques in 2014. 27ubsequently, it was reported that AIM2 could contribute to chronic inflammation by promoting the release of proinflammatory cytokines, and AIM2 expressions were correlated with plaque sizes. 280][31] Our results showed AIM2 was an independent risk factor for CAD, which is consistent with the finding by Pan et al. 28 Our study also found that AIM2 was positively correlated with the neutrophil count, NLR, MLR, PLR, and SII, suggesting that AIM2 is possibly involved in the inflammatory process in CAD.The expressions of ASC and Caspase-1 (AIM2 infalmmasome), GSDMD (the pyroptosis marker), and IL-1β and IL-18 (the proinflammatory factors) were all significantly upregulated in the CAD group, that further indicated the chronic inflammatory mechanism mediated by AIM2 inflammasome.
We treated THP-1 monocytes, primary monocytes, and their derived macrophages with AIM2 agonist, then found a significant increase in AIM2 inflammasome components, cell pyroptosis marker, proinflammatory cytokines and the migration and adhesion ability of THP-1 monocyte, while the opposite result was observed with AIM2 inhibitors A151 and inflammasome inhibitor JC2-11. 32yroptosis is a pattern of programmed cell death accompanied by an inflammatory response, and it plays an important role in the inflammatory response associated with CAD. 33,34We found that AIM2 overexpression could cause pyroptosis, and monocyte-macrophage death in advanced AS lesions might promote the formation of necrotic nuclei and release inflammatory cytokines, chemokines, proteases, and other substances that greatly increase the risk of plaque rupture. 35evertheless, our study still has some limitations.First, the study population size is small, and some confounding factors may influence our results.Studies have shown that some other factors were involved in regulating of AIM2 expression.For example, Oxidized Low-Density Lipoprotein (ox-LDL) can increase the expression of AIM2 through the Nuclear Factor-Κb signaling pathway. 28Primary cultured human aortic endothelial cells and human aortic smooth muscle cells show a rapid increase in endogenous AIM2 expression after exposure to TNF-α or dsDNA stimulation. 27,36Thus, other factors might also participant in regulating AIM2 expressions in monocytes, further study with multicenter large sample with extensive investigations on multiple confounding factors was required.Second, due to the less amount of monocytes in peripheral blood, our experiment was mainly performed in THP-1 monocytes.Thirdly, we only used JAK2 inhibitor to treat the cells, while the experiment with JAK2 overexpression in THP-1 cells could further verify the results.

| CONCLUSIONS
In conclusion, we suggest that IFN-γ may activate AIM2 inflammasome by the JAK2/STAT1 signaling pathway, triggering a series of subsequent inflammatory responses that promotes activation and pyroptosis in monocytes, and ultimately participate in the chronic inflammation in CAD.

F I G U R E 1
Elevated expression of AIM2 and downstream genes in PBLs of CAD patients.(A) The mRNA levels of AIM2 were detected by qPCR in the PBLs of CAD patients (n = 133) and controls (n = 123).The mRNA levels of (B) ASC, (C) Caspase-1, (D) IL-1β, and (E) IL-18 were detected by qPCR in the PBLs of partial CAD patients (n = 25) and controls (n = 25).The correlations of expressions of AIM2 and ASC (F) or Caspase-1 (G) in all subgroup participants.Significant was defined as p < 0.05.*p < 0.05; **p < 0.01; ****p < .0001.CAD, Coronary Artery Disease; PBLs, Peripheral Blood Leukocytes.

T A B L E 3
Correlation analysis and multivariate stepwise linear regression analysis of the AIM2 expression with clinical parameters of all participants.Significant was defined as p < 0.05.

F I G U R E 2
The correlations of mRNA level of AIM2 and MLR (A), NLR (B), PLR (C), and SII (D) in all participants.AIM2, Absent in Melanoma 2; CAD, Coronary Artery Disease; MLR, Monocyte-to-Lymphocyte Ratio; mRNA, messenger RNA; NLR, Neutrophil-to-Lymphocyte Ratio; PLR, Platelet-to-Lymphocyte Ratio; SII, Systemic Immune-Inflammation Index.T A B L E 4 Relationships between AIM2 mRNA expression and CAD.